Anthrax is a disease believed to be caused by the spore-forming bacterium, Bacillus anthracis (“B. anthracis”), a bacterium that is readily found in soil. B. anthracis is believed to primarily cause disease in plant-eating animals. Though infrequent, when humans do become infected, they usually acquire the bacterium from contact with infected animals, animal hides or hair, or animal feces. The human disease has a relatively short incubation period (less than a week) and usually progresses rapidly to a fatal outcome.
In humans, anthrax may occur in three different forms: cutaneous anthrax, gastrointestinal anthrax and inhalation anthrax. Cutaneous anthrax, the most common form in humans, is usually acquired when the bacterium, or spores of the bacterium, enter the body through an abrasion or cut on the skin. The bacteria multiply at the site of the abrasion, cause a local edema, and a series of skin lesions—papule, vesicle, pustule and necrotic ulcer—are sequentially produced. Lymph nodes nearby the site are eventually infected by the bacteria and, in cases where the organisms then enter the bloodstream (20% of cases), the disease is often fatal. Gastrointestinal anthrax can be caused by eating contaminated meat. Initial symptoms include nausea, vomiting and fever. Later, infected individuals present with abdominal pain, severe diarrhea and vomiting of blood. This type of anthrax is fatal in 25% to 60% of cases. Inhalation anthrax (also called woolsorters' disease) can be acquired through inhalation of the bacteria or spores. Initial symptoms are similar to those of a common cold. Symptoms then worsen and these individuals present with high fever, chest pain and breathing problems. The infection normally progresses systemically and produces a hemorrhagic pathology. Inhalation anthrax is fatal in almost 100% of cases. Cutaneous anthrax is acquired via injured skin or membranes, entry sites where the spore germinate into vegetative cells. Proliferation of vegetative cells results in gelatinous edema. Alternatively, inhalation of the spores results in high fever and chest pain. Both types may be fatal unless the invasive aspect of the infection may be intercepted.
B. anthracis is able to form highly resistant spores that can survive in the environment for prolonged periods of time. Only the spore form of B. anthracis is believed to be infectious, the vegetative form of the organism has not been shown to be transmittable. The vegetative bacilli are believed to survive very poorly outside the host. In fact, it is believed that the complete B. anthracis life cycle may solely occur within the mammalian host. Once B. anthracis spores enter the body, they are phagocytosed by macrophages. The incubation period of B. anthracis spores within the human body can be up to 60 days prior to germination. Not only do spores survive within the macrophage, but it is believed that they germinate within the macrophage phagosomal compartment. The macrophages are also believed to serve as a vehicle for transporting the bacteria to regional lymph nodes, particularly the mediastinal lymph nodes, where escape from macrophages allows their entry into the bloodstream. B. anthracis expression of a toxin causes macrophage lysis, and allows bacteria to enter the bloodstream. Vegetative bacterial counts can reach up to 108 per milliliter of blood. Once germination has occurred within the body, the bacteria remain in their vegetative form, with sporulation being suppressed in the absence of air.
B. anthracis is believed to possess two major virulence components. The first virulence component is a polysaccharide capsule which contains poly-D-glutamate polypeptide. The poly-D-glutamate capsule is not believed to be toxic, but plays an important role in protecting the bacterium against anti-bacterial components of serum and phagocytic engulfment. As the B. anthracis bacterium multiplies in the host, it produces a secreted toxin which is the second virulence component of the organism. This anthrax toxin mediates symptoms of the disease in humans. Full virulence of B. anthracis is believed to require the production of a protein capsule and two toxins, namely the lethal toxin and the edema toxin. Strains lacking any one of these virulence factors are attenuated. However, the diseases caused by B. anthracis are believed to be toxin mediated, with the injection of both anthrax toxins being able to reproduce anthrax disease progression in animals. This further accentuates the need for early diagnosis and treatment as elimination of vegetative bacilli may not improve patient prognosis if high levels of toxins are already present in the bloodstream, as evidenced in animal studies. The factors essential for B. anthracis virulence all function in some manner to evade or suppress the host immune system.
The anthrax toxin is believed to comprise three distinct proteins encoded by the bacterium: protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the component of the anthrax toxin that is believed to bind to host cells using an unidentified cell-surface receptor. Once it binds to cell surfaces, EF or LF may subsequently interact with the bound PA. The complexes are then internalized by the host cell with significant effects. EF is an adenylate cyclase which causes deregulation of cellular physiology, resulting in edema. LF is a metalloprotease that cleaves specific signal transduction molecules within the cell (MAP kinase isoforms), causing deregulation of said pathways, and cell death. Injection of PA, LF or EF alone, or LF in combination with EF, into experimental animals produces no effects. However, injection of PA plus EF produces edema. Injection of PA plus LF is lethal, as is injection of PA plus EF plus LF.
As an acute, febrile disease of virtually all warm-blooded animals, including man, anthrax can be used in biological weapons (BW). For example, ten grams of anthrax spore may kill as many people as a ton of the chemical warfare agent, sarin. Terrorists have included dry spores in letters. Biological weapons of mass destruction have been developed that contain large quantities of anthrax spores for release over enemy territory. Once released, spores may contaminate a wide geographical area, infecting nearly all susceptible mammals. Due to the spore's resistance to heat and dry conditions, contaminated land may remain a danger for years. In view of the serious threat posed by the disease, effective diagnostic tools are needed to assist in prevention and control of natural and man-made outbreaks. Due to the highly lethal nature of anthrax and BW agents in general, there is great need for the development of sensitive and rapid BW agent detection. Current detection technology for biological warfare agents have traditionally relied on time-consuming laboratory analysis or onset of illness among people exposed to the BW agent.
Bacteriophages specific for B. anthracis and related B. cereus bacteria strains may be isolated and used to detect and treat these bacteria. Bacteriophages near B. anthracis spores during spore germination may be used to infect and lyse the bacteria. A variety of phage-based bacterial therapies have been reviewed. D. H. Duckworth, P. A. Gulig, “Bacteriophages: Potential treatment for bacterial infections,” BioDrugs, 16(1), 57-62 (2002). There are various environmental bacteriophages present in soils that may infect and lyse B. anthracis under controlled conditions. H. W. Ackermann, et al., “New Bacillus bacteriophage species,” Archives of Virology, 135(3-4), 333-344 (1994); H. W. Ackerman, M. S. Dubrow, Viruses of prokaryotes: General properties of bacteriophages, Boca Raton, Fla., CRC Press, Inc. (1989). Bacteriophages for B. anthracis may be isolated from various sources. For instance, Walter et al. report the isolation of Phages Nk, DB and MH for B. anthracis in topsoil. Walter, M H, Baker, D D, “Three Bacillus anthracis bacteriophages from topsoil,” Curr Microbiol. 2003 July; 47(1): 55-58.
The direct introduction of bacteriophages into an animal to prevent or fight diseases can be subject to certain potential difficulties. For example, both the bacteria and the phage have to be in the correct and synchronized growth cycles for the phage to attach. Additionally, the number of phages has to be calibrated to attach to the bacteria; if there are too many or too few phages, there will be either no attachment or no production of the lysing enzyme. The phage is preferably active enough to be effective. The phages may also be inhibited by many things including bacterial debris from the organism it is going to attack. Further complicating the direct use of a bacteriophage to treat bacterial infections is the possibility of immunological reactions within the subject being treated, potentially rendering the phage non-functional. The ability of bacteriophages to lyse and kill target bacterial may also be decreased by sunlight, UV light, desiccation or other conditions encountered during storage or use of a phage-containing therapeutic agent.
One promising approach to the detection and treatment of B. anthracis is the use of bacteriophage lytic enzymes as bacteriolytic agents. Bacteriophage lytic enzymes responsible for bacterial host lysis are also known as lysins. Many lysins can rapidly break down the bacterial cell wall in order to release progeny phage (Young, R. 1992. Bacteriophage lysis: mechanism and regulation. Microbial. Rev. 56:430-481). Structurally, lysins are commonly found as modular proteins with an amino terminal domain that confers the enzymatic activity for a peptidoglycan bond and a carboxy terminal domain that confers binding specificity to a carbohydrate epitope in the bacterial cell wall (Loessner, M., K. Kramer, F. Ebel, and S. Scherer. 2002. C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates. Mol. Microbiol. 44:335-349; Lopez, R., E. Garcia, P. Garcia, and J. L. Garcia. 1997. The pneumococcal cell wall degrading enzymes: a modular design to create new lysins? MicroB. Drug Resist. 3:199-211; Lopez, R., M. P. Gonzalez, E. Garcia, J. L. Garcia, and P. Garcia. 2000. Biological roles of two new murein hydrolases of Streptococcus pneumoniae representing examples of module shuffling. Res. Microbiol. 151:437-443; Sheehan, M. M., J. L. Garcia, R. Lopez, and P. Garcia. 1997. The lytic enzyme of the pnemococcal phage Dp-1: a chimeric enzyme of intergeneric origin. Mol. Microbiol. 25:717-725). Lysin are believed to provide at least one of the following enzymatic activities against a peptidoglycan substrate: muramidases, glucosaminidases, N-acetylmuramyl-L-alanine amidase and endopeptidases (Young, R. 1992. Bacteriophage lysis: mechanism and regulation. Microbiol. Rev. 56:430-481). Purified lysin from a bacteriophage can be applied exogenously to affect bacterial lysis (Loeffler, J. M., D. Nelson, and V. A. Fischetti. 2001. Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science. 294:2170-2172; Loessner, M., G. Wendlinger, and S. Scherer. 1995. Heterogeneous endolysins in Listeria monocytogenes bacteriophages: a new class of enzymes and evidence for conserved holin genes within the siphoviral lysis cassettes. Mol. Microbiol. 16:1231-1241; Loessner, M., S. K. Maier, H. Daubek-Puza, G. Wendlinger, and S. Scherer. 1997. Three Bacillus cereus bacteriophage endolysins are unrelated but reveal high homology to cell wall hydrolases from different bacilli. J. Bacteriol. 179:2845-2851; Nelson, D., L. Loomis, and V. A. Fischetti. 2001. Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Prot. Natl. Acad. Sci. USA. 98:4107-4112).
Lysins are normally very specific to the bacterial species from which the lysin derived phage was isolated (Fischetti, V. A. 2003. Novel method to control pathogenic bacteria on human mucous membranes. Ann. N.Y. Acad. Sci. 987:207-214; Fischetti, V. A. 2001. Phage antibacterials make a comeback. Nature Biotechnol. 19:734-735). Although the range of bacteria targeted by lysins is less restrictive than the corresponding bacteriophage, lysins still maintain a degree of specificity, having minimal effects on other bacteria including commensal organisms. While bacteriophage host ranges are largely restrictive, recognizing only one specific antigen on its bacterial host, phage lysins are less restrictive, recognizing a specific carbohydrate molecule common to the particular species of host bacteria.
Bacterial resistance to phage lysins is believed to be less likely to arise as compared with bacteriophage adsorption for at least two reasons: firstly, because bacterial lysis upon exposure to lysin is almost immediate, not giving bacteria much possibility for mutation and secondly, because lysins bind to highly conserved molecules in the bacterial cell wall that are under selective pressure not to mutate. This is evidenced by the lysins from S. pneumoniae phages binding to choline, an essential component on the S. pneumoniae cell wall, and a lysin, PlyC, targeting S. pyogenes by specifically binding the alternating (α1→2) and (α1→3) linked polyrhamnose backbone of surface carbohydrates. The exposure of bacteria to subinhibitory lysin concentrations and mutagenesis studies have not identified bacteria that are resistant to the action of phage lysins (Schuch, R., D. Nelson, and V. A. Fischetti. 2002. A bacteriolytic agent that detects and kills Bacillus anthracis. Nature. 418:884-888). In contrast, bacterial resistance to many antibiotics are easily identified using the techniques used above. Furthermore, the problem with lysogenic conversion is completely eliminated with phage lysins, and animal testing have determined lysins to be safe. Of course lysin dosage will need to be worked out, taking into account the specific activity of each lysin considered, the route of injection, and the nature of infection being treated. Unlike phages, the use of lysin will not be complicated by its uncontrolled multiplication in the host.
The use of highly specific phage lysins have an advantage over antibiotics in that lysin only effects targeted bacterial strains, while having minimal effect on other bacteria including commensals. This property of targeted bacteriocidal activity makes lysins suitable for development into an alternative therapeutic agent. In fact, through mouse models, lysins have successfully been applied in the elimination of S. pyogenes, S. pneumoniae and group B streptococcal colonization on mucosal surfaces, and the treatment of bacteremia caused by S. pneumoniae and a B. anthracis-like B. cereus strain (Cheng, Q., D. Nelson, S. Zhu, and V. A. Fischetti. 2005. Removal of group B streptococci colonizing the vagina and oropharynx of mice with a bacteriophage lytic enzyme. AntimicroB. Agents Chemother. 49:111-117; Jado, I., R. Lopez, E. Garcia, A. Fenoll, J. Casal, and P. Garcia. 2003. Phage lytic enzymes as therapy for antibiotic-resistant Streptococcus pneumoniae infection in a murine sepsis model. J. AntimicroB. Chemother. 52:967-973; Loeffler, J. M., D. Nelson, and V. A. Fischetti. 2001. Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science. 294:2170-2172; Loeffler, J. M., S. Djurkovic, and V. A. Fischetti. 2003. Phage lytic enzyme Cpl-1 as a novel antimicrobial for pneumococcal bacteremia. Infect. Immun. 71:6199-6204; Nelson, D., L. Loomis, and V. A. Fischetti. 2001. Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Prot. Natl. Acad. Sci. USA. 98:4107-4112; Schuch, R., D. Nelson, and V. A. Fischetti. 2002. A bacteriolytic agent that detects and kills Bacillus anthracis. Nature. 418:884-888).
There is an ongoing need for therapies and agents effective in the diagnosis and control of bacterial contamination, colonization and infection, particularly with respect to B. anthracis. In addition, compounds with bacteriocidal effects may be useful in the decontamination of bacteria on inanimate surfaces and objects. The bactiophage lytic enzymes provided herein are useful in providing agents useful in the detection, treatment and decontamination of B. anthracis and related bacteria.